Erythropoiesis, the production of red blood cells, occurs in the bone marrow under the physiological control of the hormone erythropoietin (EPO). Erythropoietin is a 34,000 dalton glycoprotein hormone which is synthesized in the kidney, circulates in the plasma, and is excreted in the urine. In response to changes in the level of oxygen in the blood and tissues, erythropoietin appears to stimulate both proliferation and differentiation of immature erythroblasts. It functions as a growth factor, stimulating the mitotic activity of erythroid progenitor cells, such as erythrocyte burst forming and colony-forming units. It also acts as a differentiation factor, triggering transformation of an erythrocyte colony-forming-unit into a proerythroblast (Erslev, A., New Eng. J. Med. 316:101-103 (1987)).
Normally, erythropoietin is found in very low concentrations in bodily fluids. However, under conditions of hypoxia, when oxygen transport to erythrocytes in reduced, the concentration of erythropoietin in the blood stream increases. For example, in patients suffering from aplastic anemia, there is an abnormally high concentration of erythropoietin in the urine.
Purified, homogeneous erythropoietin was characterized as a single peak on reverse phase high performance chromatography with a specific activity of at least 160,000 IU per absorbance unit at 280 nanometers. (Hewick, et al., U.S. Pat. No. 4,677,195). The DNA sequence encoding erythropoietin was purified and cloned to produce synthetic polypeptides with the same biochemical and immunological properties (Lin, U.S. Pat. No. 4,703,008). A recombinant erythropoietin molecule with oligosaccharides identical to those of the natural material has also been produced. (Sasaki, H. et al., J. Biol. Chem. 262:12059-12076 (1987)).
Despite the availability of purified recombinant erythropoietin, little is known concerning the mechanism of erythropoietin-induced erythroblast proliferation and differentiation. The specific interaction of erythropoietin with immature red blood cell progenitors remains to be characterized. This is due, at least in part, to the small number of surface erythropoietin receptor molecules on normal erythroblasts and on the erythroleukemia cell line. (Krantz, S. B. and E. Goldwasser, Proc. Natl. Acad. Sci. USA 81:7574-7578 (1984); Branch, D. R. et al., Blood 69:1782-1785 (1987); Mayeux, P. et al., FEBS Letters 211:229-233 (1987); Mufson, R. A. and T. G. Gesner, Blood 69:1485-1490 (1987); Sakaguchi, M et al., Biochem. Biophys. Res. Commun. 146:7-12 (1987); Sawyer, S. T. et al., Proc. Natl. Acad. Sci. USA 84:3690-3694 (1987); Sawyer, S. T. et al., J. Biol. Chem. 262:5554-5562 (1987); Todokoro, K. et al., Proc. Natl. Acad. Sci. USA 84:4126-4130 (1988)).
Cross-linked complexes between radioiodinated erythropoietin and cell surface proteins suggest that the receptor is made up of two polypeptides, one of which has a molecular weight of 85,000 Daltons and the other of 100,000 Daltons. More recently, the two crosslinked complexes have been subjected to V8 protease digestion, suggesting the two EPO-receptor polypeptides have identical peptide fragments and therefore may be products of the same or very similar genes (Sawyer, S. T. et al., J. Biol. Chem. 266:13343-13347 (1988)). Most cell surface binding studies, however, have revealed a single class of binding sites, averaging 300 to 600 per cell surface, with a K.sub.d of approximately 800 pM (Sawyer, S. T. et al., Proc. Natl. Acad. Sci USA 84:3690-3694 (1987)). EPO-responsive splenic erythroblasts, prepared from mice injected with the anemic strain (FVA) of the Friend leukemia virus, demonstrate a high and a low affinity binding site with dissociation constants of 100 pM and 800 pM, respectively (Sawyer, S. T. et al., J. Biol. Chem. 262:5554-5562 (1987)).
Mouse erythroleukemia cells, although unresponsive to erythropoietin, are a readily available source of EPO receptor. They have a single class of EPO receptor with fewer than 1000 sites per cell and a dissociation constant of 2.times.10.sup.-10 M. (Mayeux, P. et al., J. Biol. Chem. 262:13985-13990 (1987).; D'Andrea, A. et al., Cell 57:277-285 (1989)). Crosslinking studies with radioiodinated erythropoietin reveal two putative receptor polypeptides with molecular weights of 100,000 and 85,000 Daltons.
Knowledge of the mechanism of action of erythropoietin would be of great clinical benefit in treating a number of diseases in which the erythropoietin receptor may be dysfunctional. For instance, it is believed that the erythropoietin receptor is dysfunctional in individuals with Diamond Blackfan anemia, which is a congenital anemia in which the infant is profoundly anemic and requires red blood cell transfusions and steroid treatments. In primary proliferative polycythemia (polycythemia vera), the erythropoietin receptor may be dysfunctional but, in this case, it appears to be hyperactive with EPO levels at or below normal (Murphy, S., Polycythemia Vera In: Hematology 4th Edition (1990)), resulting in a disease characterized in adults by an excess of red blood cell mass. Indeed, mature erythroid precursors (BFU-E) have a greater sensitivity to EPO than more primitive precursors, which have an enhanced sensitivity to IL-3 (Dudley, J. M. et al., Br. J. Haematol. 75:188-194 (1990) and Dai, C. H. et al., J. Clin. Invest 87:391-396 (1991)). In contrast, secondary polycythemia (erythrocytosis) is caused by elevated levels of EPO, resulting from tissue hypoxia due to decreased oxygen tension in the blood supply or inappropriate EPO production induced by an underlying disease state (Erslev, A. J., Secondary Polycythemia (Erythrocytosis): Hematology 4th Edition (1990)). Furthermore, in autoimmune diseases, such as lupus and juvenile rheumatoid arthritis, antibodies to the erythropoietin receptor may account for the anemia associated with these diseases.